This proposal is intended to characterize the contributions of nitric oxide (NO) and adenosine (AD) in regulating sleep-wake dependent activity of hypocretinergic (HCRTergic also called orexinergic) system in the perifornical-lateral hypothalamic area (PF-LHA). The central hypothesis of this proposal is that the activation of HCRT neurons contributes to the local production of AD during spontaneous and sustained arousal;that AD production is mediated via NO production;and that a subsequent inhibition of HCRT neurons contributes to spontaneous and as well as homeostatic sleep. Three specific aims have been designed to systematically evaluate various components of the above overarching hypotheses. Specific aim-1 will test whether activation of HCRT neurons during arousal is central to NO production, which in turn contributes to local AD release during spontaneous waking and its accumulation during sustained arousal. We will measure levels of NO metabolites (NOx/ ) and AD in the dialysates collected from the PF-LHA to determine: a) whether NOx/ and AD levels are higher during spontaneous arousal vs. sleep and further accumulates during sleep deprivation, b) whether focal blockade of HCRT signaling in the PF-LHA during sleep deprivation attenuates production of NOx/ and AD, c) and whether NO contributes to the production of AD. Specific aim-2 will test whether NO produced during arousal/sustained arousal contributes to sleep by inhibiting HCRT neurons and that this effect is mediated via an A1 receptor- dependent adenosinergic mechanism. a) We will use local drug delivery via reverse microdialysis into the PF-LHA to determine whether NO in the PF-LHA contributes to spontaneous sleep as well as recovery sleep subsequent to sleep deprivation, and if it is mediated via adenosinergic mechanism. b) We will use extracellular unit recording in combination with drug delivery to determine the ability of NO to suppress the discharge of PF-LHA neurons and determine if this effect is mediated via adenosinergic mechanism. c) We will use double label immunohistochemistry in combination with drug delivery via a microdialysis probe into the PF-LHA to determine the ability of locally released NO in decreasing c-Fos expression in HCRT neurons in awake animals and the efficacy of A1 receptor-dependent adenosinergic mechanism in mediating this effect. Specific aim-3 will confirm the key components of proposed hypotheses in prepro-orexin knockout (HCRT KO) mice and determine whether a lack of HCRT signaling in these mice causes attenuated production of NO and AD during spontaneous/prolonged arousal, thus affecting their sleep regulation. a) We will measure NOx/ and adenosine levels in the dialysates collected from the PF-LHA of HCRT KO and its wild type littermate in spontaneous arousal as well as during sleep deprivation. b) We will also determine the sleep-promoting abilities of NO and AD in the PF-LHA in HCRT KO vs. wild type mice. The three complementary methods used in proposed studies, i.e., unit recording adjacent to the microdialysis probe, double labeling immunohistochemistry adjacent to the microdialysis probe, and the assay of the neurotransmitters/neuromodulators released in the diffusion field of the microdialysis probe used for local pharmacological manipulations provide a unique approach to understanding local interactions among nitrergic, adenosinergic, and HCRTergic systems within the PF-LHA during sleep and waking. We believe that an improved understanding of the proposed interactions and their integrated influences on sleep-wake behavior will likely contribute to new therapeutic strategies including novel insights into drug designing and/or the development of better combination therapies for the management of sleep disorders as well as diseases potentially related to the HCRTergic system that are common in general population as well as in Veterans. PUBLIC HEALTH RELEVANCE: The goal of this research program is to characterize the contributions of the nitric oxide and adenosine in the regulation of hypocretin (HCRT, also known as orexin) neurons during spontaneous sleep- wakefulness and during prolonged arousal. HCRTergic system is critically involved in the maintenance and stability of behavioral arousal and a loss of HCRT signaling is linked with symptoms of narcolepsy/cataplexy. An improved understanding of the proposed interactions and their integrated influences on sleep-wake behavior will likely contribute to new therapeutic strategies including novel insights related to designing drugs and/or the development of better combination therapies for the management of sleep disorders as well as diseases potentially related to the HCRTergic system that are common in general population as well as in Veterans.